Nonmuscle Myosin II-A is Essential for Mouse Placental and Embryonic Development In vertebrates three genes, Myh9, Myh10 and Myh14 encode three different isoforms of nonmuscle myosin II heavy chain II (NMHC II). The motor activity of nonmuscle myosin II (NM II) resides in the N-terminal globular head domain and filament formation resides in the C-terminal rod domain. Gene disruption studies in mice reveal dramatically different embryonic-lethal phenotypes upon knockout of NM II-A versus II-B. Ablation of NM II-A in mice results in lethality by E6.5 with defects in cell-cell adhesion and a failure to produce a competent visceral endoderm. Disrupted NM II-B causes later embryonic lethality between E14.5 and birth due to cardiac and neuronal development defects. Though our previous studies indicated that the functions of NM II-B can be partially replaced by II-A during development, it is unclear whether this is the case in the reverse situation. To investigate whether NM II-B can replace II-A and to uncover novel functions of NM II-A during development, we used homologous recombination to generate a mouse line (Ab*/Ab* mice) in which NM II-A is disrupted and cDNA encoding human NMHC II-B is placed under control of the II-A promoter. In addition, as a control mouse line, cDNA encoding human NM II-A is also inserted in the II-A locus (AmCh/AmCh). Our results show: first, normal homozygous Ab*/Ab* (II-B replacing II-A) embryos were found at embryonic day (E)6.5 indicating that II-B can rescue the visceral endoderm abnormalities found in the II-A null mice at E6.5, allowing gastrulation and organogenesis to occur. It also demonstrates that the lethality in II-A null mice was not due to a specific role of NM II-A but due to the decrease in total NM II in the embryo and the absence of any NM II at this early stage in the visceral endoderm. Second, Ab*/Ab* mice exhibit severe defects in the placenta, which result in embryonic lethality at E9.5-10.5, indicating a unique function for NM II-A in normal placental development. Third, in the mutant placentas, the fetal vessels failed to invade into the labyrinthine layer of the placenta, suggesting defects in cell migration. Abnormal actin organization, reduced focal adhesion formation and abnormal migratory behavior were found in the Ab*/Ab* mouse embryonic fibroblasts in culture, indicating that NM II-A has an important role during these processes that can not be replaced by II-B. Taken together, our results indicate the importance of an isoform-specific function for NM II-A in placenta development and highlight the role of NM II-A in cell migration and adhesion. Intact Nonmuscle Myosin II-A is Critical for Its Functions To further explore whether the isoform-specific roles of NM II-A and II-B are associated with their two functional domains (N terminal head and C terminal rod), we also generated two mouse lines in which the NM II-A is replaced by two chimeric NM IIs, one encoding the N-terminal motor domain of NMHC II-A fused to the C-terminal II-B rod domain (Aab/Aab mice) and one encoding the N-terminal domain of NMHC II-B fused to the C-terminal domain of II-A (Aba/Aba mice). Our results showed that Aba/Aba mice die at a similar age as Ab*/Ab* mice indicating that the N-terminal motor domains are interchangeable between II-A and II-B up to E9-10 despite differences in the kinetic properties of the motors. Aab/Aab mice survived to E12.5, with lethal defects in placentation although fetal vessels were found to invade into the labyrinthine layer of the placenta. In addition, these experiments confirmed that the C-terminal rod domain of NM II-A or II-B play vital roles in determining cellular localization. Collectively, our results highlight the importance of an intact NM II-A molecule for its functions during mid-gestation. Characterizing the Biochemical Properties of Full-length Nonmuscle Myosin IIs In vertebrates three different isoforms of nonmuscle myosin heavy chain (NMHC) II have been identified. Their encoded genes in humans are referred as Myh9, Myh10, Myh14 and their protein product are called NMHC-IIA, IIB and IIC respectively. Each heavy chain has an N-terminal globular head domain containing the actin and ATP binding sites required for motor activity and a long -helical, coiled coil C-terminal rod involved in dimerization and filament assembly. The biochemical properties of these isoforms have been studied by expression of an enzymatically active fragment, heavy meromyosin (HMM), which is a dimer containing the motor domains and a part of the rods. The resulting kinetic values may have some limitations in that recent studies showed intramolecular interaction between the rods and the heads involved in the switching off myosin II activity. To circumvent the limitations of HMM and assess additional biochemical properties, we have successfully produced 9 full length NM II proteins in the Sf9-baculovirus system. These proteins include full length wild type NM II-A, II-A with the N93K point mutation, II-A with the D1424N point mutation, II-A with the E1841K point mutation, wild type II-B, IIB with B2 insert (IIB-B2), and II-B with the R709C point mutation. Additionally, two chimeric NMHC II proteins with either the amino-terminal head of II-A fused to the rod of II-B (IIAB) or the II-B head preceding the II-A rod: (IIBA) were made. We have also made an enzymatically active NM II fragment, S1 which is shorter than HMM and is a monomer: IIB-B2 S1. Our initial purification experiments showed that in contrast to the previous utility of FLAG affinity chromatography in NMHC II-A HMM purification, the FLAG tag at the C-terminal of full length II-A did not aid in purification. Based on this fact, we appended the FLAG tag to the N-terminal end where it does aid in purification of these full length NM IIs. The purification and biochemical assays of these proteins are underway.